MICR3001 Past Exam Questions

2014-2018 + Example Questions

To whoever stumbled upon this document,

This is a compilation of my answers to MICR3001 past exam questions from 2014-2018 and example
questions (provided by some lecturers at the end of the lecture in semester 2 of 2019) organised by
module (and sometimes lecture). I have also included the past exams arranged by year at the very
bottom of the document.

Some of these answers are structured as essays but most have been structured in dot point form
since we were told that questions can be answered in dot points or essays as long as sufficient detail
has been provided. Relevant diagrams from lectures (and occasionally other sources) have been
included to provide a complete answer. However, please double check answers with lecture material
as I cannot guarantee these are 100% correct.

Note: 2019 was the first year where they changed the exam structure to six sections where you
could choose to answer one of two questions in each section.

I hope this helps someone and good luck for the exam!
- Isini
Module 1: Bacterial Pathogenesis
(a) Write about the virulence mechanisms employed by Streptococcus pneumoniae to exert
its pathogenic effect AND discuss the implications for vaccination. (2018, 2015, 2014)

Capsule
The capsule is essential to the virulence of S. pneumoniae, and strains that do not express capsules
are avirulent. It forms a physical barrier and is highly charged at physiological pH which prevents the
engulfment of pneumococci from alveolar macrophages and other phagocytic cells. For each
capsular polysaccharide (CPS) type, virulence is proportional to CPS thickness.

Vaccines
Vaccine development for S. pneumoniae has focused on the use of CPS as an antigen and several
vaccines are available including the 23V, 7V and 13V. The 23V vaccine contains antigens from the 23
most common CPS types. Immunisation with this vaccine elicits an immune response against the CPS
antigens and the production of antibodies that protect the individual against future infections by this
organism. However, the 23V vaccine is only effectively used in older age groups since it is poorly
immunogenic in infants (<2years old). The 7V vaccine is covalently conjugated to a protein carrier
and this conjugation dramatically increases the immunogenicity of CPS antigens. Therefore, the 7V
vaccine is highly immunogenic in infants but only protects against 7 serotypes. The 13V vaccine is a
second-generation conjugated vaccine which protects against 6 new serotypes and has replaced the
7V vaccine in most countries. Since conjugated vaccines are only effective against some serotypes,
these vaccines have caused a reduction in vaccine types and replacement by potentially invasive
non-vaccine types.

Capsule transformation
S. pneumoniae readily exchange cps loci in a process known as capsular transformation. This occurs
often via recombination based on vaccine selective pressures. For instance, consider a person
infected with both S. pneumoniae with Type 19A CPS and with S. pneumoniae with Type 19F CPS
who is given a vaccine that is effective against 19F (but not 19A). If a S. pneumoniae cell with Type
19A capsule lyses and releases its genetic material, the type-specific region of the cps locus of this
lysed cell may be taken up by the other strain of S. pneumoniae with the Type 19F CPS and
incorporated into its genome through genetic recombination. Therefore, although the majority of
the 19F CPS S. pneumoniae were killed, a small subgroup would have taken up 19A CPS DNA
allowing the original 19F serotype to produce a 19A CPS and therefore change capsule (and
serotype). Therefore, vaccination against specific serotypes has led to an increase in non-vaccine
serotypes.

Other virulence factors

Immunostimulatory factors released by LytA
LytA, an autolysin, degrades peptidoglycan which causes cell lysis and the release of pneumococcal
antigens such as pneumolysin, peptidoglycan, and teichoic acids. Pneumolysin binds cholesterol
which forms pores in the host membrane leading to cell lysis. It is cytotoxic to alveolar and
endothelial cells and inhibits the activity of ciliated cells. Pneumolysin also activates the classical
complement pathway (by binding the Fc component of antibodies) and stimulates monocytes to
produce cytokines. Additionally, the cell wall components, teichoic acid and peptidoglycan, are
potent immunostimulatory molecules which also activate complement and induce the production of
cytokines (IL-1, TNFα). Thus, these cell wall components and pneumolysin drive an expanding
inflammatory response which damages lung tissue and leads to leakage of fluid from blood vessels,
creating the fluid in the lung that is characteristic of pneumonia.

Adherence, colonisation and entry into lung

Choline-binding proteins (PspA, PspC) and pili aid in the attachment of S. pneumoniae to epithelial
cells. PspA and PspC prevent complement-mediated opsonisation via two different mechanisms.
PspA is very electronegative which can block complement binding and it also binds host lactoferrin
which protects S. pneumoniae against its bactericidal effects. On the other hand, PspC binds the
complement regulatory protein factor H to mediate resistance to complement by preventing the
formation of C3b. S. pneumoniae also has two types of pili which aid in adherence to epithelial cells
and stimulates cytokine production.

PavA and enolase aid in the attachment of S. pneumoniae to the extracellular matrix. PavA binds
fibronectin and assists with adherence to host cells while enolase binds plasminogen. Neuraminidase
is also expressed by S. pneumoniae and removes terminal sugars from human glycoproteins. For
example, neuraminidase removes the sialic acid from lactoferrin which reduces its bactericidal
activity.

(b) Antigenic variation of bacterial surface antigens has important implications for both
bacterial pathogenesis and vaccine development. Using a diagram, describe the molecular
mechanism mediating antigenic variation in the Neisseria gonorrhoeae type IV pili, and
outline the implications of this process for bacterial pathogenesis. (2018, 2016)
The gonococcal chromosome has one copy of the complete pilin gene, called the pilE locus, which
encodes the structural pilin protein that polymerises to form the type IV pili found in N.
gonorrhoeae. The chromosome also contains up to 11 copies of variant-encoding pilin genes which
lack promoter elements meaning transcription is not observed. These silent, non-functional copies
are known as pilS loci. Antigenic variation of the pilE locus is mediated by unidirectional
recombination with DNA from one or more of the pilS loci. All pil genes contain variable pil gene
segments (mini-cassettes) interspersed with short conserved regions. The Neisseria pilin protein
contains 6 variable mini-cassettes (mc) which result in significant antigenic variation.

(arrow = promoter element of pilE, white rectangles = conserved regions of pilE and pilS, Sma-cla = DNA sequence that is
involved in pilin recombination)

The pilin proteins that form antigenic variants of the pili are conserved for the N terminus but vary at
the remaining C terminus which is exposed at the surface of the pili. Therefore, recombination
results in the creation of new antigenically different Type IV pili meaning antibodies against pili
encoded by the old pilE gene won't work on pili based on the new pilE variant. Pili were first thought
to be an ideal vaccine candidate since they are immunogenic, are expressed by all strains and are
essential for colonisation. However, due to the significant antigenic variation of pilin in N.
gonorrhoeae, the vaccine was found to be ineffective against different strains or reinfection.

(c) Phase variation of bacterial surface antigens has important implications for both bacterial
pathogenesis and vaccine development. Using a diagram, describe the molecular
mechanism mediating phase variation in the Neisseria opa gene and outline the
implications of this process for bacterial pathogenesis. (2015)

Molecular mechanism

Phase variation refers to the high frequency switching “on and off” of genes encoding bacterial
surface antigens, such as the colony opacity associated (opa) gene in N. gonorrhoeae. N.
gonorrhoeae has multiple copies of complete opa genes scattered throughout its genome and these
genes encode several, antigenically distinct Opa proteins. Phase variation in N. gonorrhoeae occurs
when the number of 5’-CTCTT-3’ tandem repeat units is altered through ‘slipped strand mispairing’
(strand misalignment during normal DNA replication and repair). This gain or loss of pentameric
repeat units in opa genes causes a frame shift mutation in the Opa open reading frame and
determines whether an intact protein can be made. For instance, when there are 6 repeats, a
functional Opa protein is produced however 5 or 7 repeats results in no protein since translation
may end early.

Slipped strand mispairing can also occur in the promotor element of a gene which changes crucial
spatial relationships between promoter elements leading to altered RNA polymerase binding
efficiency and altered gene expression.

Implications for bacterial pathogenesis

The random and independent switching of opa genes on and off, either in the open reading frame or
at the promoter, is an alternative random mechanism for generating diversity on the bacterial
surface. For instance, phase variation allows the existence of bacteria expressing no Opa proteins,
one Opa protein or bacteria expressing multiple Opa proteins simultaneously. For n number of
genes, there would be 2n bacterial phenotypes. Additionally, the frequency of this reversible
switching is very high (up to 1% per generation) especially considering bacteria have a high
replication rate. Firstly, phase variation results in the ability to evade the immune system since
variants in the bacterial population may be selected for. This would result in the proliferation of
selected variants and enhanced overall survival. Secondly, phase variation allows the expression of
the ideal bacterial phenotype for a particular microenvironment within the host
(d) Neisseria meningitidis is an important human pathogen that causes significant morbidity
and mortality. Discuss the virulence factors that enable Neisseria meningitidis to cause
disease and the vaccines used to prevent infections caused by this pathogen. (2018, 2017)

Virulence factors

Capsule
● Protects against phagocytic killing, opsonisation and complement-mediated bactericidal
killing in the blood → ↑ survival in blood
● Poorly immunogenic → does not usually elicit a protective immune response (probably why
capsular antigen-based vaccine is conjugated)
Adhesins
● Colonisation: initial attachment to host cells via pili and more intimate contact via Opc & Opa
proteins. Subepithelial space is reservoir.
● Opa & Opc:
o Variable in number expressed, size and antigenicity
o Opa binds CD66, Opc binds heparin (highly sulphated glycosaminoglycan)

LPS
● G -ve so LPS in outer membrane
● Cell lyses or releases outer membrane blebs of LPS → LPS binds host LBP (LPS binding
protein) →LPS-LBP binds CD14 on macrophages/neutrophils/dendritic cells → Engages TLR4
system → cytokine release (TNFα, Interferon-γ, IL-1, Il-8) → coagulation pathway → blood
clotting, inflammation → vasculitis, multiple organ failure & death

Porins
● Outer membrane proteins (PorA, PorB)
● Function unknown
● Immunogenic → reacts with antibodies → enhance immune response

Iron acquisition proteins

● Acquire iron from hosts. Majority of host iron present as:
o Haemoglobin (intracellular)
o Transferrin and lactoferrin (extracellular)
Vaccine
● Two effective conjugated vaccines available (based on capsular antigen):
o C
 o A, C, Y, W-135.

● NEW Boxsero vaccine for serogroup B

o Serogroup B capsules has structures identical to those on human cells → serogroup
B-based polysaccharide vaccine could lead to antibodies against our own proteins
o Reverse vaccinology used to make Boxsero
o Based on outer membrane proteins

(e) Write about the virulence mechanisms employed by uropathogenic Escherichia coli to
exert its pathogenic effect AND discuss the implications for both treatment and
vaccination. (2017, 2016, 2015, 2014)
Virulence factors
These virulence factors may occur together on “pathogenicity islands” on the E. coli chromosome.
Fimbriae
Type 1 fimbriae
● Binds to D-mannose containing glycoproteins in the bladder via fimH adhesin
● Associated with bladder colonisation (cystitis)
● Type 1 fimbriae are expressed in IBCs, which subvert host responses and may lead to
bacterial persistence.
● Phase variable via site specific inversion to regulate the expression of pili from fimA (major
subunit) to fimH (adhesin)
o Invertible DNA element, (fim
switch), contains the promoter for
genes encoding structural
fimbriae subunits
o Promoter is properly orientated
for transcription when the
inverted element is in the ON
orientation
o DNA inversion is mediated by site-
specific recombinases fimB and fimE
o fimB turns ON whereas fimE turns OFF

P fimbriae
● Bind to the Gal-Gal disaccharide in the upper urinary tract via PapG tip adhesin
● Associated with kidney colonisation (remember ‘P’ for pyelonephritis)
● Phase variable via epigenetic switching
o Promoter for P-fimbriae is under epigenetic regulation.
 o Methylation of the GATC site near papBA promotes RNA polymerase binding
allowing easier transcription → ON
o Methylations of the GATC site near papl causes transition to OFF

Auto-transporter Adhesins
● Antigen 43: expression causes bacterial clumping → biofilm formation, IBC formation →
persistence in the bladder
Toxins
● Hemolysin (HlyA): pore-forming toxin that inserts into host cell membrane → cell lysis,
apoptosis, cytokine production
● Cytotoxic necrotizing factor 1 (CNF-1): kills epithelial cells (via membrane ruffling, actin
rearrangement)
● Secreted autotransporter serine protease (Sat): Causes vacuolation and tissue damage

Other

Cell surface polysaccharides

Capsule and O-antigen mediate serum resistance and promote host evasion.
● O antigen of LPS
o Required for survival in human serum
o Inhibits complement-mediated killing.
● Capsule
o Resistance to killing by host immune cells

Iron acquisition systems: multiple systems (e.g. aerobactin which scavenges for iron that the bacteria
need to grow)

Implications for both treatment and vaccination.

Current treatment: heavily reliant on antibiotics and there is no vaccine to prevent UTI

Antibiotics
Problem: antibiotic resistance and multidrug resistant UPEC

Specific antibiotic used depends on if it is short term (uncomplicated UTI), long term (complicated
UTI) or if catheter infection
 ● First line, alternative, last line antibiotics
● Catheter infections are treated with broad spectrum antibiotics since hospitals can have man
different bacteria that can cause UTI

Vaccines
NO vaccine currently

Difficult to target capsular polysaccharides and O-antigen due to high diversity and the limited
immunogenicity

In animal trials: vaccines based on fimbriae since these are highly immunogenic
● Type 1 fimbriae: Tip associated FimH adhesin
● P fimbriae: Tip associated PapG adhesin
Problems: Phase variable

Mannosides:
● MOA: inhibit FimH adhesin of UPEC → prevent UTI by blocking adhesion
● Novel treatment that seems to be more effective in preventing acute UTI from UPEC than
antibiotics (in mice)

(f) Discuss “shigellosis”, naming the microbe responsible, the main symptoms of disease and
THREE (3) defining microbiological features. Describe the virulence determining “main
players” and how these effector molecules contribute to cell invasion and cell-to-cell
spread. (2014)

Shigellosis

Microbe responsible: Shigella sp.

Main symptoms:

● Mucoid and bloody stools, characteristic of bacillary dysentery.

o Shigella invades intestinal epithelial cells → very destructive so blood is intestinal
lining
● Fever, severe abdominal cramps
● Watery diarrhoea

THREE (3) defining microbiological features

● Non motile, non-spore forming, rod shaped, facultative anaerobic (with or without O2)
bacteria
● Restricted host range – Humans and some nonhuman primates.
● Is an invasive pathogen:
• Shigella leaves lumen of gut and enters epithelial cells

Virulence determining “main players”

The main virulence determining players at the invasion proteins (IpaB, IpaC, IpaD) and the motility
proteins (IcsA, host actin).

Invasion proteins
The invasion proteins work in conjunction with the type 3 secretion system (T3SS). IpaB/D complex
forms a molecular “plug” at the end of the T3SS syringe to stop effector molecules leaking out when
not needed. Upon contact with a host cell, IpaD dissociates, and IpaB and IpaC form a pore in the
membrane. Effector molecules are secreted that cause cell cytoskeleton rearrangement, and uptake
of the bacterium (i.e. cause non-phagocytic cell to become phagocytic and internalise the bacteria).

Once inside the epithelial cells, the organisms escape the phagosomal vesicle through IpaB and IpaC
proteins which are secreted by T3SS and are inserted into the membrane of the phagocytic vesicle.
They then enter the cytoplasm where they multiply.

Effector molecules secreted by T3SS induce a dramatic immune response which leads to shedding of
intestinal lining and aids in bacterial entry (migration of leukocytes through mucosa leads to separation of tight junctions between
columnar epithelial cells).

Note: shigella may enter via the basolateral side of epithelial cells or enter M cells (which randomly samples stuff in lumen) to avoid apical
brush border of epithelial cells that have villi.

Motility proteins

Shigella is technically a non-motile bacterium but they can move within host cells via IcsA and host
actin. After escaping phagosome into cytoplasm, IcsA accumulates at the pole of the bacterium. IcsA
induces polymerisation of host cell actin at one pole of the rod-shaped bacteria which propels them
forward. This motility can be used to invade adjacent cells by pushing against the cell membrane.
 (g) Recent evidence demonstrates that at approximately 96 hours following internalization by
macrophages, Mycobacterium tuberculosis can breach the phagosomal membrane and gain
access to the cytoplasm. Describe the critical effector molecule that enables this AND discuss
the promising vaccine candidate currently under development that is exploiting this
property (2018, 2017, 2016, 2015)

Pathogenesis

 Transmission: person-to-person spread via aerosols (even over long distances)

 1/3 of world infected! But not everyone has symptoms (90-95%)
 Pathogenesis
1. Alveolar macrophages phagocytose Mtb
2. Inhibition of phagosome-lysosome fusion: A
glycolipid in Mtb cell wall (LAM) and the secretion
of PknG inhibits fusion of phagosome and
lysosome → preventing lysis
3. Escape into cytoplasm: Mtb escape the
phagosome through ESAT-6 (a pore-forming toxin)
which is secreted through a Type 4 Secretion
System (ESX)
4. ∴ Mtb ‘hides’ in macrophages and do not provoke
a humoral immune response (no Ab by B cells) and
replicates safely within macrophages
5. More macrophages enter and together to form a
mature granuloma to wall off infection and
caseous necrosis occurs in the centre (inhospitable, hypoxic). Mtb may remain dormant
for years inside granuloma with no symptoms (latent TB)
6. But immunosuppression/age can cause dormant bacteria to become active, replicate in
macrophages and spread into lungs & other organs (active TB)
  Mycolic acid cell wall makes it very hardy → resists weak disinfectant and can survive on dry
surfaces

ESAT-6 is secreted by ESC (T4SS) and is a pore-forming toxin that perforates phagosome membrane
allowing Mycobacterium tuberculosis (Mtb) escape into the cytoplasm.

Vaccine

● BCG – original live, attenuated vaccine

o Problems: not effective for preventing adult TB, fails to stimulate the right kind of
immune response
● Recombinant BCG (BCG ΔureC::hly)
o Instead of ESAT-6 secretion, BCG ΔureC::hly secrets pore-forming listeriolysin which
breaks down the phagosome
o Replace urease C gene with listeriolysin gene from listeria
▪ Urease C normally prevents acidification of phagosome → inhibits
phagolysosomal maturation → ↑ survival of Mtb in macrophage
▪ Urease C deficient strain would:
● Reduce intraphagosomal pH → promote phagolysosome fusion
● Provides the optimal pH for listeriolysin stability
▪ Listeriolysin results in the release of antigens and bacterial DNA into the
cytosol → then PEST sequence targets listeriolysin for proteasomal
degradation (once it reaches the cytoplasm)
∴ No urease C means Mtb is DEGRADED and listeriolysin means antigens are released into
cytoplasm.

BCG vaccine leads to phagosome-lysosome fusion and presentation of antigens on MHCII molecules
which prime a CD4 T cell response

BCG ΔureC::hly causes the rupture of phagolysosomes (through listeriolysin) and some antigens to
end up in the cytoplasm (mimicking real infection). These cytosolic antigens cause antigens to be
presented on MHCI molecules to prime a CD8 cytotoxic T cell response (which is very important
since Mtb live intracellularly). This enhanced T cell response means that recombinant BCG ureC::hly
is more effective than parental BCG.
10 Reverse vaccinology_13.8_Walker

Lecturer

1. Using a diagram, the strategy used by Novartis to identify Chlamydia trachomatis antigens that
promote a CD4-Th1 immune response.
Problem: chlamydia grows intracellularly, and it is therefore difficult for antibodies to initiate
an immune response (through CD4-Th1).
Two forms
1. Elementary body → non-replicative infectious particle
2. Reticulate body → non-infectious intracellular replicative form

1. Mice were infected with Chlamydia EB (Elementary Bodies).

2. Spleens were then harvested post-infection.
3. Spleen cells were stimulated with EB ex vivo.
4. CD4+ Th1 cells were cloned from single cells in tissue culture.
5. CD4+ Th1 cells were then tested by adoptive transfer into naive mice, to identify
which CD4+ Th1 clones provided protective immunity from Chlamydia challenge (e.g.
see slide 9 for how protection would be assessed using mouse models).
6. Using reverse vaccinology procedures, Chlamydia genes were expressed in E. coli then
individually purified.
7. In vivo stimulation with each individual Chlamydia protein was used to identify which
of these Chlamydia proteins the protective CD4+ Th1 cell clone reacts with.
8. Antigens that are recognized by protective CD4+ Th1 cell clones are used to formulate
a vaccine.

2. Using a diagram, describe the three-step strategy employed by Novartis to develop an

experimental group A Streptococcal vaccine.
See below

Streptococcal vaccine.
(h) Using diagrams, describe the three-step strategy employed by Novartis to develop an
experimental group A streptococcal vaccine. Outline why this is a more efficient approach to
antigen identification in comparison to reverse vaccinology (2017, 2015, 2014)

Antigen identification

Since antigens eliciting protective antibody responses are usually

- secreted toxins

- highly expressed, surface-exposed components

Focus on finding these classes of proteins

Mining bacterial surfaceome for antigens

1. Analysing surfaceome
Protease (trypsin) cleaves surface proteins and these peptides are isolated
LC-MS/MS is used to identify peptides
2. Assess antibody binding
Flow cytometry was used to validate the presence of proteins on the GAS surface using
antibodies raised against each recombinant surface protein
3. Assess immunogenicity
Through protein microarray were screened for immunity in vivo through the use of
sera from GAS-infected human patients
Why is this antigen selection process better than reverse vaccinology?

 Large number of candidates needs to go through in vivo and in vitro screening to identify
very few antigens conferring protections
 This biological testing is labour-intensive, expensive and time-consuming → bottleneck in
vaccine discovery process
 Pre-screening strategies that reduce the number of antigens that have to undergo biological
testing would substantially shorten time needed to identify the final vaccine formulation.
 (a) Describe with the aid of a diagram how to construct a live vaccinia virus vector expressing a
recombinant rabies virus antigen. (2018, 2017, 2016, 2014)

1) Clone gene encoding rabies antigen

2) Transfer antigen gene to a vaccinia virus (VV) shuttle vector
a. The thymidine kinase (TK) gene of vaccinia virus is nonessential for growth of the
virus in tissue culture + TK deletion attenuates virus in humans (good)
b. So, add a TK gene with large internal deletion into E. coli plasmid
c. Add a VV promoter in front of gene encoding antigen
d. Insert gene and VV promotor within large internal deletion of TK gene
Gene insertion is done through restriction enzymes (which cut DNA) and DNA ligase
(which joins DNA)
3) The plasmid (containing cloned TK gene + foreign gene insert) is transfected into eukaryotic
cells infected by wild-type VV
4) Homologous recombination occurs between TK gene of vaccinia virus and TK flanked
sequences in plasmid (at a high enough freq. that a reasonable no. of progeny have
incorporated gene of interest)
5) Collect VV from eukaryote cell line
6) Select for viruses that are TK− (i.e. TK gene is disrupted by gene of interest) by growing virus
in the presence of bromodeoxyuridine (BUdR).
a. Since TK converts BUdR to toxic product, this process selects for VV with gene of
interest

Example Questions from Lectures (Walker):

1. Using examples, describe in detail strategies by

which bacteria may vary their cell surface
architecture (question would specify either
antigenic or phase variation).
Done above

2. Using examples, describe in detail strategies by

which recombinant subunit vaccines may be
used to prevent infection.

Example: recombinant pertussis vaccine

Bordetella pertussis
 Causes whooping cough
 Secretes the pertussis toxin
 If you change 2 amino acids in enzymatic S1
subunit of pertussis toxin → create pertussis
toxoid which doesn't have toxic activity
  This toxoid can be a recombinant subunit vaccine since it is not toxic but will elicit a immune
response
 Since toxin → toxoid is such a small change (2AAs), use site-directed mutagenesis

Method: Site -directed mutagenesis

1) DNA coding sequence of normal gene is cloned
into plasmid vector
2) Separate two strands of DNA to isolate ssDNA
template
3) Short DNA oligonucleotide is synthesised that is
complementary to the region of the gene to be
mutated except for the single base change in the
appropriate codon (X)
4) Oligonucleotide is annealed to the ssDNA form of
cloned sequence
5) Oligonucleotide acts as a primer for DNA
polymerase which will replicate DNA to create
dsDNA
6) dsDNA is used to transform E. coli and screen for
mutant plasmid (since plasmid is not always taken up)
7) Incorporate mutation into B. pertussis chromosome using double-crossover homologous
recombination system
8) B. pertussis now produces toxoid (with amino acid changes) instead of toxin. Toxoid then
can be purified to make into vaccine

3. Using examples, describe in detail strategies by which recombinant vaccines based on

vaccinia virus are produced and used to prevent infectious diseases.

4. Using a diagram, describe the 3-step strategy employed by Novartis to identify Chlamydia
trachomatis antigens that promote a CD4-Th1 immune response.
Module 2: Viral Pathogenesis
a) Influenza viruses are an important cause of respiratory infection in humans and also infect
other host species such as pigs and birds. Describe how influenza virus can evolve in avian
hosts and cross the species barrier into the human population. How could this spill-over of
avian viruses into the human population be prevented? Once established in the human
population, influenza virus can continue to evolve via the process of antigenic drift. Define
antigenic drift and state how this is different to antigenic shift. (2018)
a. Variant Q: Influenza viruses are an important cause of respiratory infection in
humans and also infect other host species such as pigs and birds. The ongoing
epidemic and pandemic threat of influenza virus infection to the human population
is primarily caused by antigenic change and escape of the virus from immune
mediated protection. Discuss the different mechanisms leading to these changes
and provide an overview of the dynamics of infection and transmission between
these different hosts. (2014)

Evolution in avian hosts

Low pathogenic avian influenza (LPAI) can evolve into highly pathogenic avian influenza (HPAI) in
poultry.
● LPAI has precursor hemagglutinin (HAO) which must be cleaved by *specific enzymes* in
order to be activated to HA. These enzymes are only present in respiratory and digestive
tracts → therefore, localised and mild infection
● HPAI has a mutated cleavage site (AAs next to site) which can be cleaved by proteases
present throughout the body → therefore, systemic & deadly infection

Crossing species-barrier
Pandemics caused by Influenza A (e.g. Spanish flu) usually occur through the reassortment between
the genomes of human and animal strains (e.g. avian, swine) which results in new strains in humans.
Antigenic shift occurs when a host cell (e.g. bird)
is co-infected with different influenza types (e.g.
avian & human). Influenza viruses have a
segmented RNA genome, so, when they both
undergo replication and capsid packaging, RNA
segments can be mispackaged into another virus
through reassortment. This creates antigenically
different progeny that have a new HA or NA
glycoprotein that has never been exposed to a
human immune system → entire population
susceptible → pandemic.

RAPID change

Prevention of avian virus spill-over of into human population

● Reducing human-poultry contact e.g. shutting down live poultry markets in Asia/Africa (this
worked in Hong Kong)
 ● However, in some areas, shutting down live poultry markets may not be effective since it
may still occur through illicit means (which is even more difficult to trace). In these cases,
educating those who handle poultry is important. For example,
o Know that sick poultry can cause illness in humans through contact
o Be able to identify sick poultry, report to authorities and slaughter
o PPE + proper cleaning when handling, cleaning cages daily

Evolution in humans

Antigenic drift (and occasional antigenic shift) is the reason why we continually get epidemics of flu
yearly and why flu vaccines from this season may not work next flu season.

In humans, influenza can evolve through antigenic drift. Antigenic drift occurs when the HA (or NA)
gene acquires spontaneous mutations during replication which lead to changes in the antigenic
nature of these glycoproteins. Mutants that escape immune surveillance are selected for.

*Also, influenza viruses are RNA viruses which have polymerases with poor proof-reading ability →
this means virus can insert mutations easily.

GRADUAL but continuous change

31 Influenza_16.10_Short
Lecturer Q
Why is H5N1 considered a potential pandemic threat? What strategies could help reduce the risk of
it becoming a pandemic? What strategies could be used to mitigate disease severity if it did emerge
as a pandemic?

Potential pandemic threat

 Highly pathogenic avian influenza (HAPI e.g. H5N1) is able to cause systemic infection unlike
low pathogenic avian influenza (LAPI)
o Causes severe disease with HIGH MORTALITY, much higher than usual (animal
influenza infecting humans aren’t usually this bad)
o Influenza has the antigens hemagglutinin (HA) and neuraminidase (NA) on its surface
o Precursor HA (HAO) requires cleavage by a trypsin-like enzymes to be activated into
HA. These enzymes are present in respiratory and digestive tracts. This leads to
localised infection
o HAPI has a slightly mutated cleavage site in HAO allowing many proteases in the body
to cleave and activate HAO to HA causing systemic infection
 Influenza can infect poultry
o Particularly, in n a number of countries where poultry and human are in close contact
o People have lots of contact with poultry (esp. in poultry markets) and consume poultry.
o Very difficult to eliminate
 LAPI → HAPI mutation (e.g. H5N1) occurs in poultry
o Unusual but problematic tract of H5N1
 Influenza has a segmented genome → able to undergo antigenic shift
 o This occurs when two virus type con-infect a host cell and DNA from both ends up
being packaged into virus progeny → creating new virus type
 Causes respiratory infection → very hard to contain
 Underlying medical condition (that are more prevalent in 21 st century) can increase risk of
infection

Reduce risk of becoming a pandemic

 Reduce opportunities for human exposure to the largest reservoir of the virus: infected
poultry and other infected animals.
o Rapid detection of poultry outbreaks
o Emergency introduction of control measures, including the destruction all infected
or exposed poultry stock
o Proper disposal of carcasses
o Shutting down live poultry markets in Asia/Africa (this worked in Hong Kong) and
massive culling of birds
o However, in some areas, shutting down live poultry markets & mass culling may not
be effective since it may still occur through illicit means (which is even more difficult
to trace). In these cases, educating those who handle poultry is important. E.g.
 Know that sick poultry can cause illness in humans through contact
 Be able to identify sick poultry, report to authorities and slaughter
 PPE + proper cleaning when handling, cleaning cages daily
Strategies to mitigate disease severity if pandemic

 Surveillance of people in airports → thermal imaging

 Healthcare professionals with proper PPE to prevent transmission between patients
 Protect risk groups → maybe give neuraminidase inhibitors (oseltamivir and zanamivir) to
some (like babies who may die if infected) when symptoms first appear
b) How do herpesviruses persist in their hosts? Compare and contrast one example each for
alpha, beta and gamma-herpesviruses. (2018)
Herpes viruses can cause lytic infections, as well as latent infections which allow them to persist in
hosts.

α-HV: Neurotrophic
Example: Herpes Simplex Virus-1 (HSV-1)
Latency establish in trigeminal ganglia following primary infection.
See below Q for details.

γ-HV: Lymphotropic
Example: Epstein-Barr Virus (EBV)
EBV usually infects epithelial cells, B cells
Primary infection can be asymptomatic or cause mononucleosis (‘kissing disease’) especially in
adolescents.

Saliva (e.g. kissing) → enter orally → infect epithelium → infect tonsillar B cells, transform them and
spread through B cell proliferation (B cells go to lymph nodes all over body + spleen).

EBV can cause lytic and latent (mostly B cells) infections. EBV establishes latency in B cells
(reservoirs) and drives B cell proliferation. Although EBV progeny are not made in latently infected
cells, a small subset of the viral genome is still transcribed (all the viral latency genes). By restricting
expression of its genome, EBV also reduces recognition by antigen-specific lymphocytes.

B cells undergoing reactivation can release virus which can then go on to infect epithelial cells which
leads to periodic viral shedding in hosts. Reactivation is most common in immune-deficient
individuals.
Latent EBV infections are associated with lymphomas. Especially in immunosuppressed individuals
where the reduction of cell-mediated immune response may lead to uncontrolled EBV-driven B-cell
lymphoproliferation → lymphoma.

β-HV
Example: Human cytomegalovirus (HCMV)
Not sure how HCMV enters but from murine CMV studies, it is probably nasally, where it targets
olfactory neurons.

Nasal neuroepithelium → olfactory neurons → lymph nodes to infect dendritic cells (+ other immune
cells) → dendritic cells migrate from lymph nodes back to the blood → systemic

HCMV can persist in myeloid progenitor cells (in bone marrow), and following migration to organs,
these cells can undergo terminal differentiation leading to reactivation of latent infection.
Reactivation can cause symptomatic or asymptomatic disease.

c) Human herpes virus 1 & 2 (herpes simplex virus types 1 & 2) and human herpes virus 3
(varicella zoster virus) share the ability to establish persistence in their host. Discuss the
mechanisms of this persistence and describe the disease consequences for each of these
viruses. (2017)
α-HV: HSV-1. HSV-2, VSV
Herpes viruses can cause lytic infections, as well as latent infections which allow them to persist in
hosts. Herpesviruses focus on long-term transmission meaning they select against very high
virulence (disease consequences, esp. α-herpesviruses is usually not very severe).

HSV
HSV-1 commonly causes oral lesions while HSV-1 commonly causes genital lesions through a similar
mechanism.
*However, both oral and genital lesions can be a result of either HSV-1 and HSV-2.

HSV-1
HSV-1 mostly infects epithelial cells and neurons.
It causes lytic infections (replicates and infects neighbouring cells) in most cells and latent infections
in neurons.

HSV-1 predominantly enters through nasal neuroepithelial cells (according to mouse studies) where
it causes lytic infection (replicates and infects neighbouring cells). It can also enter orally and through broken skin,
replicate at entry and make its way up to trigeminal ganglia via a sensory branch of trigeminal but nasal neuroepithelium is what was
mentioned in lecture.
Primary infection: Nasal neuroepithelial cells → olfactory neuron → trigeminal ganglia …latent…
reactivation: → goes out down either ophthalmic/maxillary/mandibular branches of trigeminal n. →
perioral lesions usually (if it goes up ophthalmic branch → keratitis)

Primary infection: When HSV-1 enters sensory nerves

(it is neurotropic), it travels up its axon to the cell
body (retrograde transport) where it begins its latent
infection. Sensory neurons of the face have their cell
bodies within the trigeminal ganglia, and this
becomes the reservoir of latent HSV-1.

Reactivation: HSV-1 may occasionally send some viral

copies down the axon of the sensory nerve to infect
epithelial cells (anterograde transport). The lesions
that result from epithelial infection can appear again
and again throughout a person’s life (triggered by
stress, sunlight, etc.).

If the virus reactivates by travelling from ganglion towards CNS (quite rare) → fatal encephalitis (see
CNS lecture, HSV-1 is ↓ neuroinvasive but ↑
neurovirulence)

HSV-2
The pathogenesis of HSV-2 is very similar to HSV-1
but it more commonly causes genital lesions/blisters
(by establishing latency in the sacral ganglia) and is
typically transmitted sexually.
It can also cause severe neonatal illness.

Varicella-Zoster Virus (VSV)

Aerosols → infect respiratory epithelial cells → infect
circulating T cells → lymphatic/blood spread (associated with fever, malaise, headache)
→ T cells infect epithelial cells of the skin & mucosa → chickenpox lesions

Latent infection are established in peripheral ganglia (e.g. trigeminal & dorsal root ganglia). They get
there through retrograde transport from skin via sensory nerves like HSV or if leukocytes infect
ganglia directly.

Primary infection causes chickenpox. Reactivation causes shingles in the same way HSV causes
lesions (anterograde transport down sensory nerve to cause chickenpox skin lesion).
 d) Variant Q: Herpes viruses comprise a broad group of human pathogens that share the
common ability to establish persistence in their host. Discuss the mechanisms and
consequences of persistence for two herpes viruses. (2015)
See above

Probably HSV-1 & EBV

e) The World Health Organisation has plans to eradicate the viral disease polio from the world
within the decade. Do you think this is possible? Explain your answer using relevant
information about the disease, the virus and its mode of transmission and strategies of
prevention and control. Your arguments should also refer to the current epidemiological
picture of polio outbreaks and any major obstacles that may hinder this objective of the
WHO. (2014)

22 Vector-borne viruses, Hall, 11.9:

Lecturer Qs

1) Define an arbovirus and list the common types of vectors.

Arboviruses are arthropod-borne viruses that are transmitted to a vertebrate host and are
able to replicate in both the host and the vector.

2) List three medically important arboviruses in Australasia, describe their transmission cycles
and the diseases they cause.

Transmission cycle
West Nile Virus (WNV)

● The Kunjin strain of West Nile occurs in Australasia and PNG - transmitted by Culex
mosquitoes

● Mosquito bite → WNV infects skin cells → infects immune cells → infected immune cells get
into lymph nodes → blood → asymptomatic OR develop West Nile fever
o Flu like symptoms, fever
● Sometimes enters the CNS (neuroinvasive) → acute meningitis, encephalitis, and/or
profound muscle weakness (peripheral nerves)
o Mosquito bite → viremia → cross BBB → enters cerebral blood vessels → infects
endothelial cells → leaves through basal side to infect underlying brain tissue →
encephalitis/meningitis

Ross River Virus (RRV)

Asymptomatic infection in children.

● Arthritis (debilitating symptom of RRV) in ankles, fingers, knees & wrists

o Mosquito bite → … → RRV infects synovial cells & macrophages in joints →
inflammatory cell infiltrate and mediators released → arthritis
● Fever
● Rash on trunk

Dengue

1. Classical Dengue

Young kids get mild version, older children + adults get debilitating disease

● ‘Break-bone fever’ → Severe headaches, muscle/back/joint pain

● Acute febrile illness
● Vomiting & diarrhea
2. Severe dengue (Dengue haemorrhagic fever/Dengue shock syndrome)
● Less common
● Leakiness of small blood vessels → haemorrhaging → shock (possibly fatal)
A strong risk factor for severe dengue, is pre-existing suboptimal immunity which leads to antibody
(Ab)-dependent enhancement of infection (see next Q for mechanism).

3) Dengue is an important medical pathogen of global significance. Despite billions of dollars

spent to develop control measures, there is still no vaccine available. Discuss this with
respect to the mechanisms by which dengue infection produces disease. Include in your
answer a suitable strategy for developing a dengue vaccine and explain the rationale

Enhanced infection (causing fatal dengue haemorrhagic

fever/shock syndrome) can be caused when antibodies to one
serotype enhance infection when person is infected with a
different serotype.

Antibody-dependent enhancement

① Person infected with Type 1 dengue → infects

macrophages & monocytes → mild febrile illness → recovery
→ antibodies against type 1 dengue (T1 Ab)

②Reinfected with dengue but different type (Type 2 dengue)

③ T1 Ab weakly binds Type 2 dengue but not enough to

neutralise Type 2 dengue

④ However, T1 Ab-bound Type-2 dengue can bind and enter

macrophages and monocytes through Fc receptor

⑤ This means Type 2 dengue can infect through two

methods which enhances infection

⑥ ↑ Inflammatory cytokines leads to excessive, unregulated

leakiness of blood vessels → haemorrhaging

One of the main hurdles of developing a dengue vaccine is the

concern that a vaccine increases the risk of developing
immune-enhanced dengue. A vaccine for all four serotypes has been developed, but it failed since it
increased the risk of getting severe disease in dengue-naïve children.

Therefore, a dengue vaccine needs to elicit a balanced protective immune response against all four
serotypes without inducing dengue virus enhancing antibodies that could lead to severe disease in
vaccinated people.

Vaccine strategy

Recombinant subunit dengue vaccines (where dose adjustments can allow balanced immunity
against all serotypes. But subunit vaccines are usually less immunogenic compared live, attenuated,
so maybe give multiple immunisations (according to a dosing schedule).

Alternative is give the above failed vaccine to seropositive people (i.e. find a method to quickly check
serostatus and vaccinate seropositive individuals).

4) What factors play a role in the transmission of arboviruses

Factors

● Lifespan of arthropod vectors. Must survive for long enough to obtain at least two blood
meals to become infected by and transmit arbovirus
● Virus strain
● Density of hosts (e.g. urban areas)
● Host preference: Presence of reservoir species (which may amplify virus). Many types of
hosts present.
● Vector competence: ability to transmit virus, vertebrate hosts it can infect
● Rainfall, temp, tides, humidity: affects number/quality of breeding grounds
● Global warming and climate change can potentially affect mosquito-borne disease ecology
and incidence

a) Dengue is a medically important virus, with substantial burden on global health. Describe
the transmission cycle, the diseases, the proposed pathogenesis, diagnostic tests,
treatments, preventative and control measures of dengue virus. (2018)

Transmission

Dengue is an arbovirus transmitted through the vector, Aedes Aegypti (esp. north QLD). Mosquitos
infect humans which can reinfect mosquitos. Rarely, parent mosquitos can pass on infection to
offspring through vertical transmission.

Diseases & Pathogenesis

Dengue infection can cause classical dengue or severe form of dengue (dengue haemorrhagic
fever/dengue shock syndrome) and infecting mononuclear cells is a central part of pathogenesis.

See above Q for details of disease & proposed pathogenesis

Diagnostic tests

Clinical diagnosis must be confirmed from laboratory results. Diagnosis will be made after detection
of the following:

● Identify dengue virus in clinical material

● Isolate viral RNA in clinical material
● ↑ dengue-specific antibodies in serum

A tourniquet test can measure haemorrhagic tendency → to confirm haemorrhagic dengue fever

Treatments

● No specific treatment → symptomatic treatment

 ● Fluid replacement to alleviate shock & blood transfusions in the case of dengue
haemorrhaging fever

Preventative & control measures

● Live, attenuated tetravalent vaccine failed

● Control infection by eliminating breeding sites of A. Aegypti in urban environments
● Personal protection e.g. mosquito repellent & long sleeves

b) Ross River virus disease and dengue are the most common arboviral diseases in humans in
Australia. Describe the viruses that cause these diseases, with details of 1) their transmission
cycles, 2) the disease symptoms they cause, 3) how they are diagnosed and 4) how they can
be treated and prevented. Include in your answer a brief definition of an arbovirus. (2017,
2016, 2014, 2013)

Arbovirus is an arthropod-borne virus, meaning it is transmitted through arthropod vectors (e.g.

mosquitos, ticks, sandflies).

Dengue – see above Qs

Ross River Virus (RRV)

Transmission cycles
Transmission cycles occur between humans and
mosquitos and between mosquitoes and
horses/kangaroos.

Disease symptoms
Asymptomatic infection in children
● Arthritis in ankles, fingers, knees &
wrists
o Since RRV infects synovial cells and macrophages in joints → inflammatory cells
infiltrate, mediators released
● Fever
● Rash on trunk
Diagnosis
●

Treatment & prevention

● Symptomatic treatment
o Analgesics for pain/fever, anti-inflammatories for arthritis
● Prevention: no vaccine so control measures such as:
o Personal: protective clothing, mosquito repellents, house screens
o Control vector breeding

Example paper question

What are the two arboviruses that cause most disease in Australia? Describe these viruses, with
details of their transmission cycles, the diseases they cause, how they are diagnosed and how they
can be treated and prevented. Include in your answer a brief definition of an arbovirus.

1. Ross River Virus → most common arbovirus disease in Australia

2. Dengue?
See above

23 Zoonotic Viral Diseases, Hall, 12.9

Lecturer Qs

Can email him and ask if response is good enough: roy.hall@uq.edu.au

1) Define a zoonotic viral disease and describe 3 examples.

Zootonic viruses are transmitted from animals to humans. Examples include rabies, cowpox,
arenavirus.

2) Hendra and ABL are recently discovered viruses of bats in Australia. What are the likely
factors responsible for their recent emergence as pathogens of man and domestic animals.

Both Hendra virus and Australian Bat Lyssavirus are zoonotic viruses that hosted by flying
foxes in Australia. However, Hendra virus infection is more likely obtained by humans
through close contact with an infected horse (who got infected from a flying fox) whereas
ABL infection is obtained by humans through a bite from an infected flying fox (however,
there has been a case of ABL in horses meaning there is a possibility of transmission
between horses and humans).

The emergence of new zoonotic viruses such as Hendra virus and ABL are likely a result of
the following factors:
 Encroachment of human habitation and agriculture into areas of wilderness
o Increasing population leads to urbanisation and the encroachment of human
habitation into the natural habitat. This significantly alters the habitat of
wildlife species.
o Altered behaviours of wildlife can result from this, including urban-adapted
wildlife which may carry zoonotic viruses or act as reservoir hosts
o This can lead to close proximity of humans with wildlife species which may
carry virus (e.g. humans and bats)
 Natural viruses of wild animals cross the species barrier and infect humans and
domestic animals
Influenza and SARS are examples of established animal virus which crossed the
species barrier into humans (with subsequent human-to-human transmission).
 Agricultural intensification
Livestock can become an intermediate or amplifier host (e.g. poultry in avian flu)
where pathogens can evolve and spill over into humans. Additionally, agricultural
intensification can cause bats to have more closer contact with livestock.
3) List and discuss possible strategies of preventing horses and humans from becoming
infected with Hendra, and for protecting them from the disease.

Transmission
Transmission of Hendra virus occurs between flying foxes through the exposure of birthing
fluids but flying foxes appear to be unaffected by the disease. Horses are infected through
exposure to fluids from flying foxes (which contain Hendra virus), particularly through the
consumption of spats. Spats can be contaminated with Hendra virus since they are created
when flying foxes suck out juice/nectar from fruits and spit them onto the ground and
therefore contain contaminated saliva and possibly other fluids. Horses become diseased
and show symptoms (unlike flying foxes)

Strategies to prevent infection in horses and humans focus on preventing bat-to-horse,

horse-to-human and horse-to-horse transmission.

Strategies to prevent infection in horses and humans

Note: measures designed to prevent infection in horses will also reduce infections in
humans.

Reduce transmission from bats to horses since transmission occurs when horses consume
contaminated pasture, feed or water
 Fence areas to prevent horses grazing under fruiting trees that attract bats
 Do not locate horse water troughs and food under fruiting trees

Reduce transmission of Hendra virus from sick horses to humans and between horses.

 Train personnel dealing with ill horses with correct control and emergency
procedures
o E.g. isolating sick horses from other horses, people and animals until you
have a vet’s opinion
 Ensure training in the use of Hendra virus PPE

Reduce risk of infection in horses and humans

 Vaccinate horses, vets and people who work closely with horses

Treating a potential infection in humans

 Availability of Hendra virus immunotherapy to those potentially exposed (it is in

clinical trials but will be used in an emergency

4) What measures are available to prevent or treat disease caused by infection with Australian
Bat Lyssavirus.
 Australian Bat Lyssavirus (ABL) is antigenically similar to classical rabies but can be
distinguished genetically. Therefore, antibodies to rabies virus neutralises ABL
Transmission to humans is through a bite/scratch from an infected bat.

Post-exposure therapy
1. Thoroughly wash wound immediately & apply an antiseptic solution
Seek medical attention →
2. Rabies immunoglobulin (human antibodies specific for rabies)
3. Rabies vaccine

If given soon enough, rabies immunoglobulin and vaccine can prevent development of
infection.

Pre-exposure

 Rabies vaccine can prevent both rabies and ABL and is recommended in people who
handle or come in close contact with bats in Australia (e.g. bat carers, vets, wildlife
officers) or are travelling to countries where rabies is common.

b) The respiratory and gastro-intestinal tracts are arguably the most common portals of entry
for a range of different viruses. Highlight and discuss both the common and distinct features
of viral infection at these two sites using examples of each. (2017, 2015)

a. Variant of above Q: We inhale and ingest a wide variety of viral pathogens with an
equally wide range of clinical outcomes arising from infection. Using examples,
discuss the various outcomes that can result from infection via the respiratory route
and the gastrointestinal tract (use two examples for each site). An important
respiratory pathogen is influenza virus with evolutionary change to the viral genome
underpinning its ability to cause both yearly epidemics and occasional pandemics.
Briefly explain the molecular basis of the changes to the viral genome that drives
these two outbreak scenarios. (2016)

NOT variant Q:

Respiratory route Gastrointestinal route

Transmission is usually through the inhalation Transmission is usually through the faecal-oral
of aerosols route.

Examples: Infection via this route is difficult since viruses

need to be able to resist stomach acid (which is
1. Local infection:
why many GI viruses are non-enveloped), and
a. Rhinovirus (common cold proteolytic activation
causing upper respiratory tract
infection), influenza
Examples:
2. Initial asymptomatic infection then
systemic spread: 1. Local infection:
 a. Varicella-Zoster virus a. Rotavirus (gastroenteritis)
(chickenpox/shingles) is mostly
2. Initial asymptomatic infection then
spread from aerosolised skin
generalised spread (due to invasion of
lesions (respiratory epithelium
underlying tissue):
→ tonsils → infect T cells →
blood (viremia) but within T a. Polio (infects gut epithelium →
cells → skin → lesions) not lymphatics → blood (viremia)
resp. secretion → CNS to affect lower motor
neurons → paralysis)

Common

● Both can cause either local or systemic infection

● Both infect epithelial cells (either respiratory or gut)

21 Viral infections of the CNS_10.9_Hall
Lecturer Qs

1) Describe the major routes that viruses use to enter the CNS, and list an example virus for
each route. Also discuss the various disease states that viral infection of the CNS may cause.

Blood-borne invasion of the CNS

① Blood-CSF route → take advantage of weakened BBB around choroid plexus

Viruses can enter the CNS through blood vessels that supply the brain and spinal cord. The blood-
brain-barrier (BBB) prevents most viruses/bacteria/fungi from entering the CNS through tight
junctions between endothelial cells that line the blood vessels of the brain and the very dense
basement membrane. However, some viruses can enter through the choroid plexus which, unlike
the rest of the CNS, has a fenestrated endothelium and a sparse basement membrane. From here,
they can spread into the CSF of the ventricles and to the rest of the brain.

CNS invasion steps:

Virus in capillary of choroid plexus → Bypass endothelium of capillary & go into stroma → Infect
epithelium of choroid plexus → enter ventricles & spread through CSF flow → infect ependymal cells
lining ventricles → invade underlying brain tissue → encephalitis
Viruses may leave the capillary to enter the stroma of the choroid plexus through 3 methods:

● Infect endothelial cells and pass through (enteroviruses & alphas)

● Leakage through or passive transfer through fenestrations
● Trojan horse: hitchhike in a migrating monocyte or lymphocyte
(measles, mumps, HIV)

① Blood-brain-barrier

One method used by West Nile Virus to enter the CNS is by

crossing the BBB directly (maybe during BBB compromise) and
moving THROUGH endothelial cells.

Mosquito bite → viremia → cross BBB → enter cerebral blood

vessels → infect endothelial cells → leave through the basal side
→ invade underlying brain tissue → encephalitis

Neural spread to CNS

Enter sensory or motor nerve endings → retrograde axonal

transport by hitching a ride on microtubule ‘highway’ → CNS

Examples:

● Herpes viruses (Herpes simplex virus=HSV, varicella-zoster

virus=VSV, pseudo-rabies virus=PSV) enters through
sensory nerve endings
 o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
HSV1/2 usually ends up peripherally in a ganglion (e.g. trigeminal ganglion in face) and
rarely goes to towards CNS and cause encephalitis
o It just lives there near cell bodies (for life) and occasionally send down some virus
particles to cause some mucosal skin lesions (e.g. near mouth)
● Rabies through neuromuscular junction
o Infect muscle cells & replicate → transported across neuromuscular junction →
enter motor/sensory nerves
o Very short pathway through olfactory nerves but through anterograde transport
away from cell bodies towards CNS (remember people exploring caves got rabies
but no bite/scratch)

Disease States

● Encephalitis: Inflammation of the brain (encephalon)

o Viral replication in brain mattter → destructive grey matter lesions → inflammation
→ usually fatal
o Fever, drowsiness, mental confusion, convulsions, focal neurological signs
● Aseptic meningitis: inflammation of meninges
o Relatively mild disease → fever, headache, neck stiffness
● Paralysis: Infection of motor neurons → flaccid paralysis of lower limbs
● Post-infection encephalomyelitis: autoimmune attack of CNS → inflammation of brain &
spinal cord & demyelination
o Usually post-infection: symptoms similar to encephalitis
o Sometimes postvaccine: Guillan-Barre syndrome: demyelination of peripheral nerve
roots (happens post-flu vaccine)

2) Compare and contrast the terms encephalitis, meningitis and post-infectious

encephalomyelitis in the context of the pathogenesis of viral infections of the CNS.
 ● Encephalitis: Inflammation of the brain (encephalon)
o Viral replication in brain mattter → destructive grey matter lesions → inflammation
→ usually fatal
o Fever, drowsiness + more likely to cause brain dysfunction than meningitis e.g.
mental confusion, convulsions, focal neurological signs
o E.g. herpes simplex virus (HSV-1 or HSV-2)
● Aseptic meningitis: inflammation of meninges
o Relatively mild disease → fever, headache, neck stiffness
o Called aseptic meningitis when CSF test is negative after routine bacterial tests.
Usually caused by virus but can be caused by fungi or mycobacteria)
● Paralysis: Infection of motor neurons → flaccid paralysis of lower limbs
o E.g Polio
● Post-infection encephalomyelitis: autoimmune attack of CNS → inflammation of brain &
spinal cord & demyelination (but no virus found in brain/spinal cord)
o Usually post-infection: symptoms similar to encephalitis
o Sometimes postvaccine: Guillan-Barre syndrome → demyelination of peripheral
nerve roots (happens post-flu vaccine)

3) Explain the terms neuroinvasive, neurotropic and neurovirulent in the context of viral
infections.

Explain terms Context of viral infections

Neurotropic All of the below are neurotropic viruses as they infect

neural cells.
Neurotropic viruses are capable of
infecting neural cells. Rabies is highly neuroinvasive since it invades the CNS
after a bite from an animal. It is also highly neurovirulent
Neuroinvasive
since it causes almost 100% fatal encephalitis once it
Neuroinvasive viruses can enter the enters the brain.
CNS after infection of a peripheral site
Mumps is highly neuroinvasive (enters CNS after aerosol
(through hematogenous or neural
droplets) yet the immune response is effective at dealing
spread)
with infection → low neurovirulence
Neurovirulent
Herpes simplex virus has a low neuroinvasiveness of the
Neurovirulent viruses cause disease in CNS (it almost always enters the PNS). It is highly
nervous tissue → manifests as neurovirulent since if it does enter the CNS, it causes
neurological symptoms, death severe disease and may be fatal.

f) Neurological disease is a relatively uncommon consequence of virus infection. What are the
reasons for this and, using three examples, discuss how some viruses overcome the major
barriers to CNS invasion? Discuss the range of clinical consequences of viral infection in the
CNS. (2016)
 g) Viruses may gain access to various compartments of the central nervous system (CNS) and
cause disease. Describe THREE (3) different access routes that viruses may use, provide an
example of each and discuss the potential disease consequences of CNS invasion. (2015)
Lecturer Qs

11 Viral Pathogenesis_15.8_Short
1) With examples, state the factors that influence viral infection and severity

Host factors
Receptors
● HIV
o Receptor for HIV is CD4 which is found on T helper cells.
o Cofactors (CCR5) that facilitate HIV entry are occur on these cells. People with two
copies of a mutant CCR5 allele have a reduced susceptibility to HIV-1 infection. (WHO
infected)
● Rhinovirus (common cold)
o The rhinovirus receptor (ICAM-1) is found on the epithelium of the upper respiratory
tract. This means that colonisation usually affects the upper respiratory tract
(WHERE infected)
Altered Immune state
Impaired
● Organ transplantation (therapeutically acquired)
o Organ transplant recipients receive immunosuppressive drugs to prevent organ
rejection → ↓ immune response to fight infection → ↑ viral infection and ↑
severity
● HIV-AIDs
o HIV infects T helpers cells, and uses host cell to manufacture more HIV→ Both HIV-
infected and uninfected CD4 T cells die during HIV infection → ↓ T helper cells →
severely weakened immune system → ↑ viral infection and ↑ severity
Age
Severity may be increased at different ages for some viruses
● Varicella zoster virus (VZV) causes chicken pox before puberty and shingles in the elderly

Metabolic state

● Generalized malnutrition & underweight → ↑ viral infections

● Diabetes → hyperglycemic environment favours immune dysfunction (e.g., damage to the
neutrophil function, depression of the antioxidant system, and humoral immunity) → ↑
frequency and/or severity of infection

2) What would make a virus the most effective at spreading amongst the human population?

● Route of spread
o Aerosol since this does not require person-to-person contact → e.g. rhinovirus
o Human-to-human
● Animal reservoir
o Infects animals and the animal reservoir has lots of contact with people
o E.g. domestic dogs/cats
● Site of infection
 o Upper respiratory tract: this helps since more virus can be transmitted to people via
aerosols → e.g. rhinovirus
● Symptoms
o Asymptomatic infection or host is infectious before symptoms. This will delay
diagnosis and optimise spread between hosts → e.g. herpes simplex virus
o Also, medium severity illness that is difficult for the immune system to eradicate is
best since host survival is important (viruses are dependent on host) but long
infection period is also important (for maximal transmission to other hosts).
● Replication
o Best if it can replicate in all cells (e.g. binding to MHC1 cells)
● Infectious dose
o Very low infectious dose which will dramatically increase spread
● Environmental survival
o Very high survival on contaminated surfaces. For example, if viruses can survive
weeks on a benchtop, they can infect many people → e.g. rotavirus
o Survive in extreme temperatures

12 Viruses spread by the faecal-oral route (amongst others) 1_20.8_Short

Describe the pathogenesis of Rotavirus and outline strategies used to prevent infection.
① Pathogenesis
● Rotavirus is non-enveloped enteric virus → resistant to stomach acid like other enteric
viruses
1. Transmitted by the faecal-oral route
2. Infects tip of villi in small intestine
3. Replicates within gut epithelial cells
4. Infected cells cause ↑ virus particle release into gut
5. Infected cells are also damaged → digestion/absorption mechanisms don’t work → water
collects in lumen →
6. Lose water, salt → SEVERE DIARRHEA
7. Lots of virus in faeces → ready to transmit to next unfortunate person

This is a self-limiting infection BUT severe diarrhea causes lots of deaths of infants in developing
countries through dehydration.

Blue = characteristics that are shared by other enteric/gastrointestinal viruses (e.g. norovirus,
rotavirus and enteroviruses: polio+nonpolio,)

② Strategies to prevent infection

Prevent infection by vaccine but also by practicing good hygiene and having good sewerage system
(make sure drinking water does not have faecal matter)

Vaccine
First vaccine: RotaShield: live, attenuated vaccine. BAD since it caused Intussusception
(small intestine collapses inward on itself like an old-fashioned telescope)

Rotavirus-induced gastroenteritis is vaccine preventable!!

Two current vaccines: both are given orally and are live (good for gut immunity) and neither has
been associated with intussusception. Both are safe & efficacious
● RotaTeq is a live, pentavalent, human–bovine reassortant vaccine
 o Enough of a human virus to get a immune response but also enough of a bovine
virus to prevent replication in humans
o Since it is live, don’t give to severely immunocompromised children since they may
get infected.
● RotaTrix is a live, monovalent, human attenuated vaccine
o Rotarix is ideal since ↓ doses and can dose child earlier than RotaTeq vaccine
13 Viruses spread by the faecal-oral route (amongst others) 2_21.8_Short
a) The World Health Organisation has plans to eradicate the viral disease polio from the world
within the decade. Do you think this is possible? Explain your answer using relevant
information about the disease, the virus and its mode of transmission and strategies of
prevention and control. Your arguments should also refer to the current epidemiological
picture of polio outbreaks and any major obstacles that may hinder this objective of the
WHO. (2014)
Possible by may be difficult
 Since 1988, the Global Polio Eradication Initiative has reduced polio worldwide by
99% but polio is still endemic to Afghanistan, India, Nigeria and Pakistan.
Barriers
 Conflict
o People are unable to go in and vaccinate children
 Resistance to vaccination – reports of occurrence of polio in children in India who
have been previously vaccinated (> 4 doses) of oral polio vaccine
o Need herd immunity to protect these individuals
 Vaccine derived polio
o Since polio vaccine is an attenuated version of an enterovirus, the weakened
vaccine virus replicates in the intestines for a limited period allowing
immunity to be developed through antibodies.
o During this time, vaccine-virus is also excreted
o IN areas of inadequate sanitation (which is the case with the remaining four
countries), this excreted vaccine-virus can spread in the immediate
community (which may might be good since it can offer protection to other
children through passive immunisation) before dying out.
o BUT, rarely, in an under-immunised population, the excreted vaccine-virus
can continue to circulate. If the vaccine-virus circulates for a long enough
time (>12 months), it may develop mutations such that it is able to paralyse
(circulating vaccine-derived poliovirus = cVDPV)
o cVDPV has been reduced by changing the vaccine from trivalent to bivalent
(eliminating type 2 since it no longer exists)
o It can also be improved by a very effective and rapid vaccine campaigns
 Polio is an enterovirus that are transmitted via the faecal-oral route
o Easy transmission via contaminated water supply
 Does not present with easily recognised symptoms
o An infected person can remain unnoticed yet still spread virus to others.\
o But smallpox, on the other hand, has a short time between time of exposure
and initial appearance of symptoms so disease cannot usually spread far
without being noticed

Why it can be prevented

 Polio cannot infect animals
o It is a human specific disease meaning it does not infect animal reservoirs
like rabies & flu does (these are very hard to eliminate)
 o Even if the entire population was immunised against something like rabies, it
could still re-emerge if human immunity ever waned after circulating in the
animal reservoir
 Good, effective vaccine
o Polio vaccine is able to induce long-lasting immunity so that even if non-
vaccinated people get infected, immunised people should never get virus
again (flu, common cold is like this)
o But need to prevent vaccine-derived polio
 Current cases in Afghanistan, Nigeria and Pakistan are generally due to low
immunisation rates and sanitation
o We can fix this! Not impossible!


Lecturer Q
Using polio as example, describe what is necessary to eradicate a viral disease from the human
population
Above

Module 3: Fungal Pathogenesis

NOTE all dimorphic fungal pathogens (Histoplasma, Blastomyces, Coccidiomyces)

● Cause same TB-like symptoms (mild resp. infection, serious pulmonary infection,
disseminated infection with involvement of skin)
● Are geographically restricted
● Are contracted through inhalation of some sort of spore (not really person-to-person
spread).

a) What is the cause of Chicago disease? In your answer include a description of the geography,
symptoms and the infectious particle associated with this disease. (2015)

● Cause & infectious particle

Blastomycosis is the infection which is caused by:

Blastomyces dermatitidis

Blastomyces gilchristii

● Geography

Central & Southeast USA, parts of Africa, India

● Symptoms

Mild (flu-like) respiratory infection

Serious pulmonary infection

Disseminated infection with common skin involvement

● Form

Thermally dimorphic→ adopts yeast form in tissues

b) Name the five most well-known dimorphic fungal pathogens of humans found in the
Americas. Which of these causes Valley Fever? In your answer include a description of the
geography, symptoms and the infectious particle associated with this disease. (2014)

● Five most well-known dimorphic fungal pathogens of humans in Americas

● Cause & infectious particle

Coccidiomycosis is the infection which is caused by:

Coccidioides immitis

Coccidioides posadasii

● Geography
o South-east USA
o Central & South America
● Symptoms

Mild (flu-like) respiratory infection

Serious pulmonary infection

Disseminated infection with common skin & CNS involvement

● Form & how it is contracted

Dimorphic (NOT temperature dependent)

o Environment (soil)→ mould

o Tissues→ spherule (contains hundreds of endospores) which propagates
infections when released

So breathe in spores (esp. in sand storms), which then form spherules in human
host.

Most virulent known fungus→ if exposed, you will probably be infected

● Most common in which populations?

o Pregnant women
o Dark-skinned individuals
o Immunocompromised
 c) You are asked to isolate the pathogen responsible for histoplasmosis from an environmental
sample. What is the organism responsible, how would you complete your task, and what are
the risks associated? (2014)

● Cause & infectious particle

Histoplasmosis is the infection caused by: Histoplasma capsulatum

● How to isolate from environmental sample

1. Collect soil (e.g. cave)
2. Resuspend in saline solution
3. Inject Mouse
4. Wait for mouse to die
5. Harvest fungus (at 37deg)
● Symptoms

Mild flu-like respiratory infection

Serious pulmonary infection

Disseminated (often fatal) infection, can include skin

BUT if you are healthy, mostly a self-limiting disease. Severe infection mostly in
immunocompromised

16 Candida albicans & Antifungals Fungal Pathogenesis_27.8_Fraser

d) What is the most common fungal disease of humans? Describe the major features of the
main causative agent the types of infections that are commonly encountered, and the type
of treatment employed. Are there other species that have similar clinical manifestations?
Does treatment of these infections differ? (2014)

● Most common fungal disease & main causative agent

Candidiasis→ Candida albicans

● Major features
o Diploid yeast
o NO SEXUAL CYCLE
o Changes form in response to environment (quorum sensing) & other yeast
(e.g. repressed growth/hyphae if culture is growing to saturation). Can grow
as:
▪ Yeast: round→ ovoid shape, separate readily from each other
▪ Pseudohyphae: Elongated yeast cells that are attached to each other.
BRANCHING PATTERN growth
▪ True hyphal cells: mould, no obvious constrictions between cells
● Types of infections
 o In NORMAL, healthy hosts, it infects MUCOSAL surfaces causing CUTANEOUS
infections
▪ Oral thrush
▪ Vaginitis
▪ Nappy rash
▪ Plus other body parts: nail, skin, oesophageal infections & UTIs
o In IMMUNOCOMPROMISED patients

Disseminated infection

▪ How is it acquired + risk factors

Often nosocomial, NOT inhaled

Risk factors

▪ Readily forms BIOFILMS on catheters

▪ Esp. after broad-spectrum antibiotic treatment since bacteria
competition is absent
▪ Immunocompromised
▪ Stages of systemic infection
▪ Epithelial penetration (filamentous form)
▪ Vascular dissemination (yeast)
▪ Endothelial colonisation & penetration
▪ Adhesion & colonisation
● Treatments

Candida albicans can be treated with

o Systemic infections→ amphotericin B, echinocandin, fluconazole

o Oral→ nystatin
o Skin & genital yeast infections→ azoles
● Other species with similar clinical manifestations & effects on treatment

e) Describe the link between chocolate production and life-threatening fungal infections.
Provide information on the species involved, the type of disease it causes, and the treatment
employed to combat it. (2016)

● Species

Candida krusei is involved in chocolate production and is also an emerging fungal

nosocomial pathogen primarily found in the immunocompromised.

o Chocolate production

Cacao beans have to be fermented to remove bitter taste & break them
down
 This is down by Candida krusei

● Type of disease

Candidiasis

o In NORMAL, healthy hosts, it infects MUCOSAL surfaces causing CUTANEOUS

infections
▪ Oral thrush
▪ Vaginitis
▪ Nappy rash
▪ Plus other body parts: nail, skin, oesophageal infections & UTIs

Systemic candidiasis is caused by: C. albicans, C. glabrata, C. parapsilosis, C.

tropicalis, and C. krusei.

o In IMMUNOCOMPROMISED patients

Disseminated infection in seriously immunocompromised patients

▪ How is it acquired + risk factors

Often nosocomial, NOT inhaled

Risk factors

▪ Readily forms BIOFILMS on catheters

▪ Esp. after broad-spectrum antibiotic treatment since bacteria
competition is absent
▪ Immunocompromised
▪ Stages of systemic infection
▪ Epithelial penetration (filamentous form)
▪ Vascular dissemination (yeast)
▪ Endothelial colonisation & penetration
▪ Adhesion & colonisation
● Treatments

Candida albicans can be treated with

o Systemic infections→ amphotericin B, echinocandin, fluconazole

o Oral→ nystatin
o Skin & genital yeast infections→ azoles

Candida krusei is naturally resistant to fluconazole

o So treat with amphotericin B and echinocandins (caspofungin)

f) Name five classes of antifungal agents. To which class does flucytosine belong? Describe
what flucytosine is and its mechanism of action. (2018)

● Five classes of antifungal agents

1. Polyenes: Cell membrane ergosterol binding and lysis (Amphotericin B)
2. Azoles: Inhibit P450-dependent ergosterol production (Fluconazole)
3. Echinocandins: Cell wall β-1,3 glucan synthase inhibitors (Caspofungin)
4. Nucleoside analogues (Flucytosine)
5. Allylamines (Terbinafine)
● Flucytosine

o Class & What is it?

Class: Nucleoside analogue

Flucytosine is a pyramidine analogue

o MOA
1. Given as a prodrug
2. Prodrug converted by a fungal enzyme not present in humans
(cytosine deaminase) into fluorouracil
3. Fluorouracil is cytotoxic and interferes with DNA synthesis
▪ by inhibiting thymidylate synthase

g) Variant Q: Name five classes of antifungal agents. To which does UK-49858 belong? Describe
what it is and what it does. (2016)

Classes as above

● UK-49858
o Class & What is it?

Fluconazole was originally reported as UK-49858

Class: Azole (it is a triazole)

o MOA

Inhibits ergosterol synthesis→ ergosterol makes up fungal cell membrane,

therefore azoles cause

1. Azoles bind iron atom of haem group of cytochrome P450 lanosterol

14 α demethylase which inactivates enzyme
2. This increases membraine permeability and inhibits fungal growth
since ergosterols makes up fungal cell membrane.
 a. Describe the role of fluorocytosine in the treatment of fungal infections. (2015)

b. Name five classes of antifungal agents. To which does AmBisome belong? Describe
what it is and what it does. (2017) AmBisome only mentioned in lect18

● Class & What is it?

Class: polyene

AmBisome is a liposomal formulation of amphotericin B (less side effects)

● MOA
1. Polyenes bind ergosterol
2. Forms artificial pores

c. Name five classes of antifungal agents. To which class does Diflucan belong?

d. Describe what Diflucan is and what it does 2013

h) In August 2018, the media reported the first case in Australia of an emerging fungal
pathogen that has appeared in a number of countries over the last decade. What is this
organism, and what makes it so difficult to deal with? (2018)

● Organism: Candida auris

● What makes it so difficult to deal with?
o Resistance to many antifungals
o Difficult to identify: since it can be misidentified as other type of fungi which
can lead to patients getting wrong treatment
o It causes serious infections→ often causes systemic infections and even death
o It is becoming more common GLOBALLY
o Nosocomially transmitted
▪ Hospitals have lots of immunocompromised people→ death

17 Aspergillus fumigatus Fungal Pathogenesis_28.8_Fraser

i) What is the most common life-threatening mould infection of humans? Describe the major
features of the main causative agent. Are there other species that have similar clinical
manifestations? Does treatment of these infections differ? (2017, 2015)

a. Variant Q: What is the most common mould species that causes life-threatening
disease of humans? Describe the major features of this species and the types of
infections that are commonly encountered. Are there other species that have similar
clinical manifestations, but require alternate treatment? (2018)

● Most common mould species that causes life-threatening disease of humans

Aspergillus fumigatus

● Major features
 o Saprophytic mould: lives on decaying organic matter→ recycles carbon &
nitrogen
● Types of infections

1. Superficial mycoses
2. Angioinvasion
3. Vascular damage
4. Invasive pulmonary aspergillosis
5. Cerebral aspergillosis
● Pathogenesis
1. Inhale conida (asexually produced fungal spore that germinate and grow into hyphae
when conditions are favourable)
2. Conida germinates to hyphae in lung tissue

Disease is caused if A. fumigatus overcomes barriers such as pulmonary

macrophages & neutrophils that attack escaped hyphae. More common in
immunocompromised.

● Other species with similar clinical manifestations

Cryptococcus

● How is this other species treated?

Cryptococcal meningoencephalitis is treated with flucytosine, amphotericin B

(combination therapy for induction)

Maintenance therapy is fluconazole

Aspergillus is often treated with azoles, with voriconazole being more useful than
fluconazole

j) Describe the clinical manifestations and epidemiology of Aspergillus infections. What would
be the most appropriate therapeutic agent to treat these types of infections? (2013)

18 Cryptococcus neoformans Fungal Pathogenesis_29.8_Fraser

k) It is becoming increasingly apparent that cryptic species are responsible for a large number
of fungal infections normally attributed to well-known, geographically-restricted species.
Provide three examples of such cryptic species. Describe them and the disease they cause.
(2016)
19 Fungal Molecular Genetics_3.9_Fraser

l) What are the Molecular Koch's Postulates? In your explanation, use Cryptococcus
neoformans in an example. (2017) also lect18
 a. Variant Q: Describe the molecular approaches used to identify whether a gene in
Cryptococcus neoformans is required during infection, and why each step is taken.
(2013)

● Molecular Koch's postulates = Set of criteria to show a gene found in a pathogen encodes a
product that contributes to the disease caused by the pathogen

1. Phenotype/property must be associated with pathogenic strain of a species

2. Specific inactivation of the gene(s) associated with suspected virulence trait should
lead to measurable loss of pathogenicity/virulence

3. Allelic replacement of mutated gene should lead to restoration of pathogenicity

4. Gene which causes virulence must be expressed during infection

5. Immunity must be protective

● Cryptococcus neoformans

1. Phenotype/property must be associated with pathogenic strain of a species

Masson-Fontana silver stain reveals melanised C. neoformans cells in paraffin

embedded brain tissue BUT staining not observed in non-pathogenic Cryptococcus
species

2. Specific inactivation of the gene(s) associated with suspected virulence trait should
lead to measurable loss of pathogenicity/virulence
Melanin-deficient mutant exhibit reduced virulence.

4. Gene which causes virulence must be expressed during infection

o Detected in infected human brain samples (proven by presents of melanin ghosts)

Module 4: Clinical Examples

a) Using a specific example, illustrate with the aid of a diagram how genomic epidemiology is
used to track hospital outbreaks. Explain the advantages of this method in comparison with
other technologies currently being employed to track hospital outbreaks. (2018, 2016, 2015,
2014)
Genomic epidemiology = applying bacterial whole genome sequencing to monitor infection
transmission in the clinical setting.
Genomics epidemiology allows us to find out where strains come from and figure out order
of transmission.

Puerperal sepsis is a postpartum bacterial infection contracted by women post-birth and can
lead to maternal and newborn morbidity. Group A streptococcus (GAS) is the infectious
cause of puerperal sepsis.

GAS serotyping is based on M protein sequence (M1, M3) or gene sequence (e.g. emm1,
emm3). GAS of serotype M28 has been associated with recent outbreaks, including the NSW
puerperal sepsis outbreak in 2010.
 1. 11 GAS isolates were recovered from vaginal swabs, urine & blood samples
2. emm typing (sequencing emm gene) was carried out using PCR amplification
Most had distinct emm types which ruled out a single source of infection.
Five had identical emm types (emm28).
3. Pulsed-field gel electrophoresis identified one of the five strains as distinct from the
others
4. Whole genome sequencing was conducted on these five isolates. Following this, SNP
analysis of the five isolates and a reference M28 strain was carried out to find out
whether these four strains are truly identical (same source of infection) or different
(multiple sources of infection)
It was found that three isolates were almost identical even though one was from a
different patient
Therefore, one transmission could have occurred
5. This data was confirmed by other epidemiological and clinical data (e.g. both isolates
were from patients at the same hospital and both infections occurred within a 2-day
window)
6. Together, this suggests either patient-to-patient or staff-to-patient transmission of
puerperal sepsis and
Information gained from genomic epidemiology (the integration of whole-genome
sequencing and epidemiological investigations) can inform hospital policy and infection
control measures.

Traditional microbial typing methods: phenotypic based (e.g. colony morphology), gel based
(e.g. pulsed-field gel electrophoresis), sequence based (e.g. emm typing).
Yeah this is what I said:
Genomic Epidemiology – Advantages over other technologies
● Greater resolution: Can distinguish between isolates that are indistinguishable by
traditional sequencing types
● Able to predict antimicrobial resistance phenotypes and identify virulence factors.
● Results from one lab can be easily compared with those around the world → global
changes can be monitored

29 Application of Genomics to GAS Epidemiology_10.10_Walker

How was genomic epidemiology used to undertake analyses of the NSW puerperal sepsis outbreak?
(or south Asia)
See above!

33 Emerging Bacterial disease_17.10_Schembri

Give an example of a recently emerged bacterial pathogen and discuss factors involved in its
emergence. Give detail on pathogen, don’t just mention it!

Mycobacterium tuberculosis

 Transmission: person-to-person spread via aerosols (even over long distances)

 1/3 of world infected! But not everyone has symptoms (90-95%)
  Pathogenesis
1. Alveolar macrophages phagocytose Mtb
2. Inhibition of phagosome-lysosome fusion: A glycolipid in Mtb cell wall (LAM) and the
secretion of PknG inhibits fusion of phagosome and lysosome → preventing lysis
3. Escape into cytoplasm: Mtb escape the phagosome through ESAT-6 (a pore-forming
toxin) which is secreted through a Type 4 Secretion System (ESX)
4. ∴ Mtb ‘hides’ in macrophages and do not provoke a humoral immune response (no Ab
by B cells) and replicates safely within macrophages
5. More macrophages enter and together to form a mature granuloma to wall off
infection and caseous necrosis occurs in the centre (inhospitable, hypoxic). Mtb may
remain dormant for years inside granuloma with no symptoms (latent TB)
6. But immunosuppression/age can cause dormant bacteria to become active, replicate in
macrophages and spread into lungs & other organs (active TB)
 Mycolic acid cell wall makes it very hardy → resists weak disinfectant and can survive on dry
surfaces

Factors contributing to emergence

 HIV/AIDS → TB is leading cause of death in HIV-positive people

o HIV and TB speed each other progress
o HIV+ people infected by TB are at high risk of developing active TB
o HIV positive people infected with TB are at high risk of developing reactivation TB
o
 Poorly managed TB programs (emergence of MDR and XDR strains)
o Poorly supervises and incomplete treatment of TB lead to the emergence of:
o Multi-drug resistant (MDR) TB which does not respond to standard treatment and
Extensively Drug Resistant (XDR) TB where resistance to 2 nd-line drugs develops
(extremely difficult to treat)
 Crowding & the movement of people to cities
o Global trade, tourism
 This is supported by the fact that at least half of TB cases are in foreign-born
people
o People travelling in planes → index patient with TB on 8hr flight spread TB to eight
people
o Especially problematic given TB spreads via aerosol (even over large distances)
 Poverty (refugees and homeless people)
o Untreated TB spreads quickly in crowded refugee camps & shelters
o Also the homeless population in developed countries house many TB cases

Sorted by Year
2018
Question 1 (25 marks)

Answer TWO (2) of the following questions:

(b) Write about the virulence mechanisms employed by Streptococcus pneumoniae to exert its pathogenic effect AND discuss
the implications for vaccination.
(c) Neisseria meningitidis is an important human pathogen that causes significant morbidity and mortality. Discuss the virulence
factors that enable Neisseria meningitidis to cause disease and the vaccines used to prevent infections caused by this
pathogen.
(d) Recent evidence demonstrates that intracellular Mycobacterium tuberculosis can breach the phagosomal membrane and
gain access to the macrophage cytoplasm. Describe the effector molecule that enables this AND discuss the promising
vaccine candidate currently under development that is exploiting this property.

Question 2 (25 marks)

Answer TWO (2) of the following questions:

h) Antigenic variation of bacterial surface antigens has important implications for both bacterial pathogenesis and vaccine
development. Using a diagram, describe the molecular mechanism mediating antigenic variation in the Neisseria
gonorrhoeae type IV pili, and outline the implications of this process for bacterial pathogenesis.
i) Describe with the aid of a diagram how to construct a live vaccinia virus vector expressing a recombinant rabies virus
antigen.
j) Using a specific example, illustrate with the aid of a diagram how genomic epidemiology is used to track hospital outbreaks.
Explain the advantages of this method in comparison with other technologies currently being employed to track hospital
outbreaks.

Question 3 (25 marks)

Answer TWO (2) of the following questions:

a) Influenza viruses are an important cause of respiratory infection in humans and also infect other host species such as pigs
and birds. Describe how influenza virus can evolve in avian hosts and cross the species barrier into the human population.
How could this spill-over of avian viruses into the human population be prevented? Once established in the human
population, influenza virus can continue to evolve via the process of antigenic drift. Define antigenic drift and state how this
is different to antigenic shift.
b) How do herpesviruses persist in their hosts? Compare and contrast one example each for alpha, beta and gamma-
herpesviruses.
c) Dengue is a medically important virus, with substantial burden on global health. Describe the transmission cycle, the
diseases, the proposed pathogenesis, diagnostic tests, treatments, preventative and control measures of dengue virus.

Question 4 (25 marks)

Answer TWO (2) of the following questions:

a) Name five classes of antifungal agents. To which class does flucytosine belong? Describe what flucytosine is and its
mechanism of action.
b) What is the most common mould species that causes life-threatening disease of humans? Describe the major features of this
species and the types of infections that are commonly encountered. Are there other species that have similar clinical
manifestations, but require alternate treatment?
c) In August 2018, the media reported the first case in Australia of an emerging fungal pathogen that has appeared in a number
of countries over the last decade. What is this organism, and what makes it so difficult to deal with?

2017
Question 1 (25 marks)

Answer TWO (2) of the following questions:

a) Write about the virulence mechanisms employed by uropathogenic Escherichia coli to exert its pathogenic effect AND
discuss the implications for both treatment and vaccination.
b) Neisseria meningitidis is an important human pathogen that causes significant morbidity and mortality. Discuss the virulence
factors that enable Neisseria meningitidis to cause disease. What vaccines are used to prevent infections caused by
Neisseria meningitidis?
 c) Recent evidence demonstrates that at approximately 96 hours following internalization by macrophages, Mycobacterium
tuberculosis can breach the phagosomal membrane and gain access to the cytoplasm. Describe the critical effector molecule
that enables this AND discuss the promising vaccine candidate currently under development that is exploiting this property

Question 2 (25 marks)

Answer TWO (2) of the following questions:

a) Phase variation of bacterial surface antigens has important implications for both bacterial pathogenesis and vaccine
development. Using a diagram, describe the molecular mechanism mediating phase variation in the Neisseria opa gene, and
outline the implications of this process for bacterial pathogenesis.
b) Describe with the aid of a diagram how to construct a live vaccinia virus vector expressing a recombinant rabies virus
antigen.
c) Using diagrams, describe the three-step strategy employed by Novartis to develop an experimental group A streptococcal
vaccine. Outline why this is a more efficient approach to antigen identification in comparison to reverse vaccinology

Question 3 (25 marks)

Answer TWO (2) of the following questions:

a) Human herpes virus 1 & 2 (herpes simplex virus types 1 & 2) and human herpes virus 3 (varicella zoster virus) share the
ability to establish persistence in their host. Discuss the mechanisms of this persistence and describe the disease
consequences for each of these viruses.
b) Ross River virus disease and dengue are the most common arboviral diseases in humans in Australia. Describe the viruses
that cause these diseases, with details of 1) their transmission cycles, 2) the disease symptoms they cause, 3) how they are
diagnosed and 4) how they can be treated and prevented. Include in your answer a brief definition of an arbovirus.
c) The respiratory and gastro-intestinal tracts are arguably the most common portals of entry for a range of different viruses.
Highlight and discuss both the common and distinct features of viral infection at these two sites using examples of each.

Question 4 (25 marks)

Answer TWO (2) of the following questions:

a) What are the Molecular Koch's Postulates? In your explanation, use Cryptococcus neoformans in an example.
b) What is the most common life-threatening mould infection of humans? Describe the major features of the main causative
agent. Are there other species that have similar clinical manifestations? Does treatment of these infections differ?
c) Name five classes of antifungal agents. To which does AmBisome belong? Describe what it is and what it does.

2016
Question 1 (25 marks)

Answer TWO (2) of the following questions:

a) Write about the virulence mechanisms employed by uropathogenic Escherichia coli to exert its pathogenic effect AND
discuss the implications for both treatment and vaccination.
b) Neisseria meningitidis is an important human pathogen that causes significant morbidity and mortality. Discuss the virulence
factors that enable Neisseria meningitidis to cause disease. What vaccines are used to prevent infections caused by Neisseria
meningitidis?
c) Recent evidence demonstrates that intracellular Mycobacterium tuberculosis can breach the phagosomal membrane and
gain access to the macrophage cytoplasm. Describe the effector molecule that enables this AND discuss the promising
vaccine candidate currently under development that is exploiting this property.

Question 2 (25 marks)

Answer TWO (2) of the following questions:

a) Variation of bacterial surface antigens has important implications for both bacterial pathogenesis and vaccine development.
Using a diagram, describe the molecular mechanism mediating antigenic variation in the Neisseria gonorrhoeae type IV pili,
and outline the implications of this process for bacterial pathogenesis.
b) With the aid of a diagram, describe how to construct a live vaccinia virus vector expressing a recombinant rabies virus
antigen.
c) Using a specific example, illustrate how genomic epidemiology is used to track hospital outbreaks. Explain the advantages of
this method in comparison with other technologies currently being employed to track hospital outbreaks.

Question 3 (25 marks)

Answer TWO (2) of the following questions:

a) Neurological disease is a relatively uncommon consequence of virus infection. What are the reasons for this and, using three
examples, discuss how some viruses overcome the major barriers to CNS invasion? Discuss the range of clinical
consequences of viral infection in the CNS.
b) What are the two arboviruses that cause most disease in Australia?
c) Describe these viruses, with details of their transmission cycles. Describe the diseases they cause, how they are diagnosed
and how they can be treated and prevented. Include in your answer a brief definition of an arbovirus.
d) We inhale and ingest a wide variety of viral pathogens with an equally wide range of clinical outcomes arising from infection.
Using examples, discuss the various outcomes that can result from infection via the respiratory route and the
gastrointestinal tract (use two examples for each site). An important respiratory pathogen is influenza virus with
evolutionary change to the viral genome underpinning its ability to cause both yearly epidemics and occasional pandemics.
Briefly explain the molecular basis of the changes to the viral genome that drives these two outbreak scenarios.

Question 4 (25 marks)

Answer TWO (2) of the following questions:

(a) It is becoming increasingly apparent that cryptic species are responsible for a large number of fungal infections normally
attributed to well-known, geographically-restricted species. Provide three examples of such cryptic species. Describe them
and the disease they cause.
(b) Describe the link between chocolate production and life-threatening fungal infections. Provide information on the species
involved, the type of disease it causes, and the treatment employed to combat it.
(c) Name five classes of antifungal agents. To which does UK-49858 belong? Describe what it is and what it does.

2015
Question 1 (Total: 25 marks)

Answer ONLY TWO (2) of the following questions.

(a) Write about the virulence mechanisms employed by Streptococcus pneumoniae to exert its pathogenic effect AND discuss
the implications for vaccination.
(b) Write about the virulence mechanisms employed by uropathogenic Escherichia coli to exert its pathogenic effect AND
discuss the implications for vaccination.
(c) Mycobacterium tuberculosis (Mtb) produces specific effector molecules to manipulate host macrophages and aid its survival.
Recent evidence demonstrates that Mtb can breach the phagosomal membrane and gain access to the cytoplasm. Describe
the effector molecule that enables this AND discuss the promising vaccine candidate, currently under development, that is
exploiting this property.

Question 2 (Total: 25 marks)

Answer ONLY TWO (2) of the following questions.

(a) Phase variation of bacterial surface antigens has important implications for both bacterial pathogenesis and vaccine
development. Using a diagram, describe the molecular mechanism mediating phase variation in the Neisseria opa gene and
outline the implications of this process for bacterial pathogenesis.
(b) Using a diagram, describe the three-step strategy employed by Novartis to develop an experimental group A streptococcal
vaccine. Outline why this is a more efficient approach to antigen identification in comparison to reverse vaccinology.
(c) Using a specific example, illustrate how genomic epidemiology is used to track hospital outbreaks. Explain the advantages of
this method in comparison with other technologies currently being employed to track hospital outbreaks.

Question 3 (Total: 25 marks)

Answer ONLY TWO (2) of the following questions.

(a) The respiratory and gastro-intestinal tracts are arguably the most common portals of entry for a range of different viruses.
Highlight and discuss both the common and distinct features of viral infection at these two sites using examples for each.
(b) Viruses may gain access to various compartments of the central nervous system (CNS) and cause disease. Describe THREE (3)
different access routes that viruses may use, provide an example of each and discuss the potential disease consequences of
CNS invasion.
(c) Herpes viruses comprise a broad group of human pathogens that share the common ability to establish persistence in their
host. Discuss the mechanisms and consequences of persistence for two herpes viruses.

Question 4 (Total: 25 marks)

Answer ONLY TWO (2) of the following questions.

(a) What is the most common life-threatening mould infection of humans? Describe the major features of the main causative
agent. Are there other species that have similar clinical manifestations? Does treatment of these infections differ?
 (b) What is the cause of Chicago disease? In your answer include a description of the geography, symptoms and the infectious
particle associated with this disease.
(c) Describe the role of fluorocytosine in the treatment of fungal infections.

2014

Question 1 (25 marks)

Answer TWO (2) of the following questions:

(a) Write about the virulence mechanisms employed by Streptococcus pneumoniae to exert its pathogenic effect AND discuss
the implications for vaccination.
(b) Write about the virulence mechanisms employed by uropathogenic Escherichia coli to exert its pathogenic effect AND
discuss the implications for vaccination.
(c) Discuss “shigellosis”, naming the microbe responsible, the main symptoms of disease and THREE (3) defining microbiological
features. Describe the virulence determining “main players” and how these effector molecules contribute to cell invasion
and cell-to-cell spread.

Question 2 (25 marks)

Answer TWO (2) of the following questions:

a) With the aid of a diagram describe how a live vaccinia virus vector expressing a recombinant rabies virus antigen can be
constructed.
b) Using a diagram, describe the three-step strategy employed by Novartis to develop an experimental group A streptococcal
vaccine. Outline why this is a more efficient approach to antigen identification in comparison to reverse vaccinology.
c) Using a specific example, illustrate how genomic epidemiology is used to track hospital outbreaks. Explain the advantages of
this method in comparison to other technologies currently being employed to track hospital outbreaks

Question 3 (25 marks)

Answer TWO (2) of the following questions:

a) Influenza viruses are an important cause of respiratory infection in humans and also infect other host species such as pigs
and birds. The ongoing epidemic and pandemic threat of influenza virus infection to the human population is primarily
caused by antigenic change and escape of the virus from immune mediated protection. Discuss the different mechanisms
leading to these changes and provide an overview of the dynamics of infection and transmission between these different
hosts.
b) Ross River virus and dengue virus are two medically important viruses of Australasia. Provide details of their transmission
and the diseases they cause, including methods of diagnosis and treatment. Also discuss options available for the prevention
and control of each disease.
c) The World Health Organisation has plans to eradicate the viral disease polio from the world within the decade. Do you think
this is possible? Explain your answer using relevant information about the disease, the virus and its mode of transmission
and strategies of prevention and control. Your arguments should also refer to the current epidemiological picture of polio
outbreaks and any major obstacles that may hinder this objective of the WHO.

Question 4 (25 marks)

Answer TWO (2) of the following questions:

a) What is the most common fungal disease of humans? Describe the major features of the main causative agent the types of
infections that are commonly encountered, and the type of treatment employed. Are there other species that have similar
clinical manifestations? Does treatment of these infections differ?
b) Name the five most well-known dimorphic fungal pathogens of humans found in the Americas. Which of these causes Valley
Fever? In your answer include a description of the geography, symptoms and the infectious particle associated with this
disease.
c) You are asked to isolate the pathogen responsible for histoplasmosis from an environmental sample. What is the organism
responsible, how would you complete your task, and what are the risks associated?